1. Tandem affinity depletion: a combination of affinity fractionation and immunoaffinity depletion allows the detection of low-abundance components in the complex proteomes of body fluids.
- Author
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Mortezai N, Harder S, Schnabel C, Moors E, Gauly M, Schlüter H, Wagener C, and Buck F
- Subjects
- Animals, Antibody Affinity immunology, Blotting, Western, Camelids, New World immunology, Carcinoembryonic Antigen blood, Carcinoembryonic Antigen immunology, Chromatography, High Pressure Liquid, Colonic Neoplasms blood, Humans, Mass Spectrometry, Proteome immunology, Proteomics instrumentation, Reproducibility of Results, Wheat Germ Agglutinins immunology, Biomarkers blood, Chromatography, Affinity methods, Proteome analysis, Proteomics methods
- Abstract
Protein biomarker discovery in the low concentration range of human body fluids requires the enrichment of the proteins of interest. Here we report on a tandem affinity strategy: In the first step, we isolated a human plasma glyco-subproteome of healthy individuals by wheat germ agglutinin (WGA) lectin affinity chromatography. In the second step, the proteins of this subproteome were used to raise antibodies in llama (Lama glama). The heavy-chain fraction of the llama antibodies was used to deplete from the WGA lectin binding fraction all proteins normally found in human plasma. In this way, we selectively enriched the glycoprotein, CEA, a known cancer marker which had been spiked into normal plasma. As a proof of concept, we applied this method to the analysis of plasma sample from colon cancer patients. We could demonstrate the selective enrichment of CEA by a factor of 600-800.
- Published
- 2010
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